Most viruses and transposons serve as effective carriers for the introduction of foreign DNA up to 11 kb into vertebrate genomes.However,their activity markedly diminishes with payloads exceeding 11 kb.Expanding the p...Most viruses and transposons serve as effective carriers for the introduction of foreign DNA up to 11 kb into vertebrate genomes.However,their activity markedly diminishes with payloads exceeding 11 kb.Expanding the payload capacity of transposons could facilitate more sophisticated cargo designs,improving the regulation of expression and minimizing mutagenic risks associated with molecular therapeutics,metabolic engineering,and transgenic animal production.In this study,we improved the Tol2 transposon by increasing protein expression levels using a translational enhancer(QBI SP163,ST)and enhanced the nuclear targeting ability using the nuclear localization protein H2B(SHT).The modified Tol2 and ST transposon efficiently integrated large DNA cargos into human cell cultures(H1299),comparable to the well-established super PiggyBac system.Furthermore,mRNA from ST and SHT showed a significant increase in transgene delivery efficiency of large DNA payloads(8 kb,14 kb,and 24 kb)into zebrafish(Danio rerio).This study presents a modified Tol2 transposon as an enhanced nonviral vector for the delivery of large DNA payloads in transgenic applications.展开更多
Transposons are sequence elements widely distributed among genomes of all three kingdoms of life, providing genomic changes and playing significant roles in genome evolution. Trichomonas vaginalis is an excellent mode...Transposons are sequence elements widely distributed among genomes of all three kingdoms of life, providing genomic changes and playing significant roles in genome evolution. Trichomonas vaginalis is an excellent model system for transposon study since its genome （- 160 Mb） has been sequenced and is composed of - 65% transposons and other repetitive elements. In this study, we primarily report the identification of Kolobok-type transposons （termed tvBac） in T. vaginalis and the results of transposase sequence analysis. We categorized 24 novel subfamilies of the Kolobok element, including one autonomous subfamily and 23 non-autonomous subfamilies. We also identified a novel H2CH motif in tvBac transposases based on multiple sequence alignment. In addition, we supposed that tvBac and Mutator transposons may have evolved independently from a common ancestor according to our phylogenetic analysis. Our results provide basic information for the understanding of the function and evolution of tvBac transposons in particular and other related transposon families in general.展开更多
Tgf2 transposase(Tgf2-TPase),a hAT transposase from goldfish,plays an important role in fish transgenic applications.Previously,the production of the recombinant Tgf2-TPase protein required rigorous fermentation at lo...Tgf2 transposase(Tgf2-TPase),a hAT transposase from goldfish,plays an important role in fish transgenic applications.Previously,the production of the recombinant Tgf2-TPase protein required rigorous fermentation at low temperatures(22℃)and early log phase induction(OD600=0.3–0.4)in Rosetta 1(DE3)Escherichia coli lines.In order to better express the Tgf2-TPase and detect its enzyme activity,83 rare codons in Tgf2-TPase were optimized and designated Tgf2-TPase^(83).The expression results showed that the soluble recombinant Tgf2-TPase83 was highly expressed at 30℃ and was inducible at an OD600 of 0.5–0.6 in the same prokaryotic expression system.After purification by affinity chromatography,Tgf2-TPase83 with codon optimization had higher enzyme activity than the Tgf2-TPase control.Comparison of different preservation methods(freezedrying at−80℃,storage in 20%-glycerol,8%-sucrose,4%-mannitol),revealed storage of Tgf2-TPase^(83) in glycerol helped to preserve its DNase digestion activity.Furthermore,size exclusion chromatography suggested that the purified Tgf2-TPase^(83) could recognize and bind to DNA probes containing a terminal inverted repeat(TIR)and a subterminal repeat(STR)sequence of the Tgf2 transposon.Overall,the results showed that optimizing the 83 codons of Tgf2 transposase can simplify the fermentation process and improve the enzyme activity.We propose that the production of the Tgf2-Tpase83 protein in a soluble and active form could provide an alternative tool for genetic modification of fish.展开更多
Protein-mediated chromatin interactions can be revealed by coupling proximity-based ligation with chromatin immunoprecipitation.However,these techniques require complex experimental procedures and millions of cells pe...Protein-mediated chromatin interactions can be revealed by coupling proximity-based ligation with chromatin immunoprecipitation.However,these techniques require complex experimental procedures and millions of cells per experiment,which limits their widespread application in life science research.Here,we develop a novel method,Hi-Tag,that identifies high-resolution,long-range chromatin interactions through transposase tagmentation and chromatin proximity ligation(with a phosphorothioate-modified linker).Hi-Tag can be implemented using as few as 100,000 cells,involving simple experimental procedures that can be completed within 1.5 days.Meanwhile,Hi-Tag is capable of using its own data to identify the binding sites of specific proteins,based on which,it can acquire accurate interaction information.Our results suggest that Hi-Tag has great potential for advancing chromatin interaction studies,particularly in the context of limited cell availability.展开更多
Transposable elements （TEs） are mobile genetic entities ubiquitously distributed in nearly all genomes. High frequency of codons ending in A/T in TEs has been previously observed in some species. In this study, the ...Transposable elements （TEs） are mobile genetic entities ubiquitously distributed in nearly all genomes. High frequency of codons ending in A/T in TEs has been previously observed in some species. In this study, the biases in nucleotide composition and codon usage of TE transposases and host nuclear genes were investigated in the AT-rich genome of Arabidopsis thaliana and the GC-rich genome of Oryza sativa. Codons ending in A/T are more frequently used by TEs compared with their host nuclear genes. A remarkable positive correlation between highly expressed nuclear genes and C/G-ending codons were detected in O. sativa （r=0.944 and 0.839, respectively, P〈0.0001） but not in A. thaliana, indicating a close association between the GC content and gene expression level in monocot species. In both species, TE codon usage biases are similar to that of weakly expressed genes. The expression and activity of TEs may be strictly controlled in plant genomes. Mutation bias and selection pressure have simultaneously acted on the TE evolution in A. thaliana and O. sativa. The consistently observed biases of nucleotide composition and codon usage of TEs may also provide a useful clue to accurately detect TE sequences in different species.展开更多
Sustained,high level transgene expression in mammalian cells is desired in many cases for studying gene functions.Traditionally,stable transgene expression has been accomplished by using retroviral or lentiviral vecto...Sustained,high level transgene expression in mammalian cells is desired in many cases for studying gene functions.Traditionally,stable transgene expression has been accomplished by using retroviral or lentiviral vectors.However,such viral vector-mediated transgene expression is often at low levels and can be reduced over time due to low copy numbers and/or chromatin remodeling repression.The piggyBac transposon has emerged as a promising nonviral vector system for efficient gene transfer into mammalian cells.Despite its inherent advantages over lentiviral and retroviral systems,piggyBac system has not been widely used,at least in part due to their limited manipulation flexibilities.Here,we seek to optimize piggyBac-mediated transgene expression and generate a more efficient,user-friendly piggyBac system.By engineering a panel of versatile piggyBac vectors and constructing recombinant adenoviruses expressing piggyBac transposase(PBase),we demonstrate that adenovirusmediated PBase expression significantly enhances the integration efficiency and expression level of transgenes in mesenchymal stem cells and osteosarcoma cells,compared to that obtained from co-transfection of the CMV-PBase plasmid.We further determine the drug selection timeline to achieve optimal stable transgene expression.Moreover,we demonstrate that the transgene copy number of piggyBac-mediated integration is approximately 10 times higher than that mediated by retroviral vectors.Using the engineered tandem expression vector,we show that three transgenes can be simultaneously expressed in a single vector with high efficiency.Thus,these results strongly suggest that the optimized piggyBac system is a valuable tool for making stable cell lines with sustained,high transgene expression.展开更多
Efficient and reliable profiling methods are essential to study epigenetics.Tn5,one of the first identified prokaryotic transposases with high DNA-binding and tagmentation efficiency,is widely adopted in different gen...Efficient and reliable profiling methods are essential to study epigenetics.Tn5,one of the first identified prokaryotic transposases with high DNA-binding and tagmentation efficiency,is widely adopted in different genomic and epigenomic protocols for high-throughputly exploring the genome and epigenome.Based on Tn5,the Assay for Transposase-Accessible Chromatin using sequencing(ATAC-seq)and the Cleavage Under Targets and Tagmentation(CUT&Tag)were developed to measure chromatin accessibility and detect DNA–protein interactions.These methodologies can be applied to large amounts of biological samples with low-input levels,such as rare tissues,embryos,and sorted single cells.However,fast and proper processing of these epigenomic data has become a bottleneck because massive data production continues to increase quickly.Furthermore,inappropriate data analysis can generate biased or misleading conclusions.Therefore,it is essential to evaluate the performance of Tn5-based ATAC-seq and CUT&Tag data processing bioinformatics tools,many of which were developed mostly for analyzing chromatin immunoprecipitation followed by sequencing(ChIP-seq)data.Here,we conducted a comprehensive benchmarking analysis to evaluate the performance of eight popular software for processing ATAC-seq and CUT&Tag data.We compared the sensitivity,specificity,and peak width distribution for both narrow-type and broad-type peak calling.We also tested the influence of the availability of control IgG input in CUT&Tag data analysis.Finally,we evaluated the differential analysis strategies commonly used for analyzing the CUT&Tag data.Our study provided comprehensive guidance for selecting bioinformatics tools and recommended analysis strategies,which were implemented into Docker/Singularity images for streamlined data analysis.展开更多
基金supported by the National Science and Technology Innovation 2030 Major Projects(2021ZD0202200)National Natural Science Foundation of China(32171090,81970264)+1 种基金Shanghai Science and Technology Commission(21ZR1482600)2023 Youth Innovation Promotion Association CAS。
文摘Most viruses and transposons serve as effective carriers for the introduction of foreign DNA up to 11 kb into vertebrate genomes.However,their activity markedly diminishes with payloads exceeding 11 kb.Expanding the payload capacity of transposons could facilitate more sophisticated cargo designs,improving the regulation of expression and minimizing mutagenic risks associated with molecular therapeutics,metabolic engineering,and transgenic animal production.In this study,we improved the Tol2 transposon by increasing protein expression levels using a translational enhancer(QBI SP163,ST)and enhanced the nuclear targeting ability using the nuclear localization protein H2B(SHT).The modified Tol2 and ST transposon efficiently integrated large DNA cargos into human cell cultures(H1299),comparable to the well-established super PiggyBac system.Furthermore,mRNA from ST and SHT showed a significant increase in transgene delivery efficiency of large DNA payloads(8 kb,14 kb,and 24 kb)into zebrafish(Danio rerio).This study presents a modified Tol2 transposon as an enhanced nonviral vector for the delivery of large DNA payloads in transgenic applications.
基金supported by grants from the National Basic Research Program(973 Program)(Nos.2006CB910401, 2006CB910403 and 2006CB910404) awarded to JY and HSthe Chinese Ministry of Science and Technology,the National Science and Technology Key Project(No. 2008ZX10004-013) awarded to JY
文摘Transposons are sequence elements widely distributed among genomes of all three kingdoms of life, providing genomic changes and playing significant roles in genome evolution. Trichomonas vaginalis is an excellent model system for transposon study since its genome （- 160 Mb） has been sequenced and is composed of - 65% transposons and other repetitive elements. In this study, we primarily report the identification of Kolobok-type transposons （termed tvBac） in T. vaginalis and the results of transposase sequence analysis. We categorized 24 novel subfamilies of the Kolobok element, including one autonomous subfamily and 23 non-autonomous subfamilies. We also identified a novel H2CH motif in tvBac transposases based on multiple sequence alignment. In addition, we supposed that tvBac and Mutator transposons may have evolved independently from a common ancestor according to our phylogenetic analysis. Our results provide basic information for the understanding of the function and evolution of tvBac transposons in particular and other related transposon families in general.
基金This work was supported by the National Science Foundation of China(31572220,31272633,31201760)the National High Technology Research and Development Program of China(863 Program)(2011AA100403)the Shanghai University Knowledge Service Platform(ZF1206).
文摘Tgf2 transposase(Tgf2-TPase),a hAT transposase from goldfish,plays an important role in fish transgenic applications.Previously,the production of the recombinant Tgf2-TPase protein required rigorous fermentation at low temperatures(22℃)and early log phase induction(OD600=0.3–0.4)in Rosetta 1(DE3)Escherichia coli lines.In order to better express the Tgf2-TPase and detect its enzyme activity,83 rare codons in Tgf2-TPase were optimized and designated Tgf2-TPase^(83).The expression results showed that the soluble recombinant Tgf2-TPase83 was highly expressed at 30℃ and was inducible at an OD600 of 0.5–0.6 in the same prokaryotic expression system.After purification by affinity chromatography,Tgf2-TPase83 with codon optimization had higher enzyme activity than the Tgf2-TPase control.Comparison of different preservation methods(freezedrying at−80℃,storage in 20%-glycerol,8%-sucrose,4%-mannitol),revealed storage of Tgf2-TPase^(83) in glycerol helped to preserve its DNase digestion activity.Furthermore,size exclusion chromatography suggested that the purified Tgf2-TPase^(83) could recognize and bind to DNA probes containing a terminal inverted repeat(TIR)and a subterminal repeat(STR)sequence of the Tgf2 transposon.Overall,the results showed that optimizing the 83 codons of Tgf2 transposase can simplify the fermentation process and improve the enzyme activity.We propose that the production of the Tgf2-Tpase83 protein in a soluble and active form could provide an alternative tool for genetic modification of fish.
基金supported by the National Natural Science Foundation of China(32221005)the Earmarked Fund for CARS(CARS-35)+1 种基金the National Natural Science Foundation of China Outstanding Youth(32125035)Major Project of Hubei Hongshan Laboratory(2021hszd003)。
文摘Protein-mediated chromatin interactions can be revealed by coupling proximity-based ligation with chromatin immunoprecipitation.However,these techniques require complex experimental procedures and millions of cells per experiment,which limits their widespread application in life science research.Here,we develop a novel method,Hi-Tag,that identifies high-resolution,long-range chromatin interactions through transposase tagmentation and chromatin proximity ligation(with a phosphorothioate-modified linker).Hi-Tag can be implemented using as few as 100,000 cells,involving simple experimental procedures that can be completed within 1.5 days.Meanwhile,Hi-Tag is capable of using its own data to identify the binding sites of specific proteins,based on which,it can acquire accurate interaction information.Our results suggest that Hi-Tag has great potential for advancing chromatin interaction studies,particularly in the context of limited cell availability.
基金supported by National Natural Science Foundation of China(Grant No.30571146)the Rice Project(04–06) of Zhejiang Province of China
文摘Transposable elements （TEs） are mobile genetic entities ubiquitously distributed in nearly all genomes. High frequency of codons ending in A/T in TEs has been previously observed in some species. In this study, the biases in nucleotide composition and codon usage of TE transposases and host nuclear genes were investigated in the AT-rich genome of Arabidopsis thaliana and the GC-rich genome of Oryza sativa. Codons ending in A/T are more frequently used by TEs compared with their host nuclear genes. A remarkable positive correlation between highly expressed nuclear genes and C/G-ending codons were detected in O. sativa （r=0.944 and 0.839, respectively, P〈0.0001） but not in A. thaliana, indicating a close association between the GC content and gene expression level in monocot species. In both species, TE codon usage biases are similar to that of weakly expressed genes. The expression and activity of TEs may be strictly controlled in plant genomes. Mutation bias and selection pressure have simultaneously acted on the TE evolution in A. thaliana and O. sativa. The consistently observed biases of nucleotide composition and codon usage of TEs may also provide a useful clue to accurately detect TE sequences in different species.
基金supported in part by research grants from the National Institutes of Health(AT004418,AR50142,and AR054381 to TCH,RCH and HHL)the National Natural Science Foundation(Grant#81202119 to XC)+1 种基金the Chicago Biomedical Consortium Catalyst Award(RRR and TCH)supported in part by The University of Chicago Core Facility Subsidy grant from the National Center for Advancing Translational Sciences(NCATS)of the National Institutes of Health through Grant Number UL1 TR000430.
文摘Sustained,high level transgene expression in mammalian cells is desired in many cases for studying gene functions.Traditionally,stable transgene expression has been accomplished by using retroviral or lentiviral vectors.However,such viral vector-mediated transgene expression is often at low levels and can be reduced over time due to low copy numbers and/or chromatin remodeling repression.The piggyBac transposon has emerged as a promising nonviral vector system for efficient gene transfer into mammalian cells.Despite its inherent advantages over lentiviral and retroviral systems,piggyBac system has not been widely used,at least in part due to their limited manipulation flexibilities.Here,we seek to optimize piggyBac-mediated transgene expression and generate a more efficient,user-friendly piggyBac system.By engineering a panel of versatile piggyBac vectors and constructing recombinant adenoviruses expressing piggyBac transposase(PBase),we demonstrate that adenovirusmediated PBase expression significantly enhances the integration efficiency and expression level of transgenes in mesenchymal stem cells and osteosarcoma cells,compared to that obtained from co-transfection of the CMV-PBase plasmid.We further determine the drug selection timeline to achieve optimal stable transgene expression.Moreover,we demonstrate that the transgene copy number of piggyBac-mediated integration is approximately 10 times higher than that mediated by retroviral vectors.Using the engineered tandem expression vector,we show that three transgenes can be simultaneously expressed in a single vector with high efficiency.Thus,these results strongly suggest that the optimized piggyBac system is a valuable tool for making stable cell lines with sustained,high transgene expression.
基金supported by the National Institutes of Health,USA(Grant Nos.R35GM142917 to Bo Zhang,U24ES026699 to Bo Zhang,R25DA027995 to Bo Zhang,R03AG070474 to Guoyan Zhao,R01NS123571 to Guoyan Zhao,U19NS130607 to Guoyan Zhao,and U24HG012070 to Bo Zhang and Guoyan Zhao)National Institutes of Health,USA.
文摘Efficient and reliable profiling methods are essential to study epigenetics.Tn5,one of the first identified prokaryotic transposases with high DNA-binding and tagmentation efficiency,is widely adopted in different genomic and epigenomic protocols for high-throughputly exploring the genome and epigenome.Based on Tn5,the Assay for Transposase-Accessible Chromatin using sequencing(ATAC-seq)and the Cleavage Under Targets and Tagmentation(CUT&Tag)were developed to measure chromatin accessibility and detect DNA–protein interactions.These methodologies can be applied to large amounts of biological samples with low-input levels,such as rare tissues,embryos,and sorted single cells.However,fast and proper processing of these epigenomic data has become a bottleneck because massive data production continues to increase quickly.Furthermore,inappropriate data analysis can generate biased or misleading conclusions.Therefore,it is essential to evaluate the performance of Tn5-based ATAC-seq and CUT&Tag data processing bioinformatics tools,many of which were developed mostly for analyzing chromatin immunoprecipitation followed by sequencing(ChIP-seq)data.Here,we conducted a comprehensive benchmarking analysis to evaluate the performance of eight popular software for processing ATAC-seq and CUT&Tag data.We compared the sensitivity,specificity,and peak width distribution for both narrow-type and broad-type peak calling.We also tested the influence of the availability of control IgG input in CUT&Tag data analysis.Finally,we evaluated the differential analysis strategies commonly used for analyzing the CUT&Tag data.Our study provided comprehensive guidance for selecting bioinformatics tools and recommended analysis strategies,which were implemented into Docker/Singularity images for streamlined data analysis.
